J Integr Plant Biol. ›› 1999, Vol. 41 ›› Issue (11): -.

• Research Articles •    

The Construction of γ-TIP Prokaryotic Expression Vector and Preparation of the γ- TIP Antibody

ZHU Mei-Jun,WANG Xue-Chen,CHEN Jia and DU Min   

Abstract: A prokaryotic expression vector, pGEX-TIP, was constructed from Arabidopsis thaliana (L.) Heynh. Employing PCR, 205 bp fragment near 3' end of γ-TIP cDNA, which has specific aquaporin activity, was amplified and cloned into pGEX-KG. Restriction endonuclease analysis and sequencing confirmed the correct construction, and 0.4 mmoL/L IPTC can induce high expression of GST-TIP fused protein which was about 50% in total of E. coli proteins. The IPTG induced E. coli was collected and ]ysed by supersonic treatment. The fusion protein was mainly recovered as an inclusion body. The expressed GST-TIP was purified by SDS-PAGE according to their molecular weight, which was about 32 kD. The purified protein was used to immune rabbits directly or was electrophoretically eluted before it was used for immunization. The highly qualified antibody for GST-TIP was obtained, which provides a very useful protein probe for the research on localization and function of aquaporins.

Key words: Aquaporin, Aquaporin antibody, Prokaryotic expression, pGEX-KG, pGEX-TIP, Arabidopsis Thaliana

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